Project Summary Alteration of membrane lipids, particularly the reduction of total phosphatidylglycerols (PGs), is a common daptomycin resistance phenotype across many species of Gram-positive bacteria. These modifications to membrane lipid content and composition can occur through direct genetic mutations in lipid biosynthetic pathways or indirect mutations in cell envelope stress response systems that regulate expression of membrane and cell wall biosynthesis genes. We have previously characterized the altered membrane lipids in daptomycin- resistant strains of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and Corynebacterium striatum and found that: i) daptomycin resistance also significantly affected non-PG lipids, and ii) the changes manifested only in membrane lipids with specific fatty acid compositions in MRSA and VRE. Lipid biosynthesis is a promising target for the development of novel therapies for the treatment of daptomycin resistance due to the lipid-dependent mechanism of daptomycin?s bactericidal action and the differences between eukaryotic and prokaryotic lipid biosynthetic pathways. However, the membrane lipidomes of Gram-positive bacteria are diverse in the variety and ratios of lipid classes present and the fatty acid compositions of those lipids. This diversity presents a challenge to finding common lipid pathways that can be exploited to disrupt daptomycin resistance, but the central pathways of lipid synthesis and metabolism are likely to be conserved across diverse daptomycin-resistant species. The long-term goals of this project are to identify the common and differential pathways in lipid biosynthesis and metabolism that contribute to daptomycin resistance among different species of Gram-positive bacteria and to identify small molecule modulators of these pathways that can reverse daptomycin resistance. In Aim 1, we will elucidate common and differential pathways in lipid metabolism and biosynthesis that are modified in a diverse collection of bacterial pathogens with daptomycin resistance. We will also examine the fatty acid-dependent nature of lipid changes in daptomycin-resistant MRSA and VRE. In Aim 2, we will modulate daptomycin resistance with small molecules targeting lipid biosynthesis and metabolism and evaluate the effects of extracellular free fatty acids on daptomycin resistance. We expect that the pathways that are conserved among diverse bacteria species with daptomycin resistance will be universal targets for modulation with small molecules, and these small molecules will improve daptomycin susceptibility by affecting the lipids and fatty acids that favor daptomycin resistance. This project will provide new fundamental understanding of the molecular alterations that contribute to daptomycin resistance and identify novel small molecule interventions that can be adapted into effective therapeutics for treating infections from multiple species of daptomycin-resistant pathogens.